Recombinant SARS-CoV nsp12 and the use of thereof and the method for producing it

Abstract

The present invention relates to a recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) non-structural protein (nsp) 12 with an RNA polymerase activity, its expression vector, its preparation method, and its use. According to the present invention, a soluble recombinant SARS-CoV nsp12 with an RdRp activity of initiating SARS-CoV genome synthesis can be over-expressed in the transformed host cells, and conveniently purified with high purity. An in vitro replication system important for studying SARS-CoV replication can be established with the purified recombinant SARS-CoV nsp12. SARS-CoV nsp12 produced by the present invention can also be used as a target for the development of anti-viral agents against SARS-CoV. In addition, materials inhibiting RNA-dependent RNA polymerase (RdRp) activity of nsp12 can be screened efficiently according to the present invention as the optimal conditions for the RdRp assay with SARS-CoV nsp12 were found.

The invention claimed is: 
     
       1. A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) nsp12 having the amino acid sequence of SEQ ID NO:1, in which nine amino acids are deleted from the N-terminus and amino acid sequences comprising several histidine residues are fused to the N-terminus. 
     
     
       2. The recombinant SARS-CoV nsp12 according to  claim 1  that is expressed by the expression vector pTrcSARSnsp12. 
     
     
       3. A composition for screening inhibitors against RNA-dependent RNA polymerase (RdRp), comprising the recombinant SARS-CoV nsp12 of  claim 1 , MnCl 2 , and NTP (nucleoside triphosphate). 
     
     
       4. The composition according to  claim 3 , wherein the recombinant SARS-CoV nsp12 is expressed by the expression vector pTrcSARSnsp 12. 
     
     
       5. The composition according to  claim 3 , wherein the NTP includes ATP, GTP, CTP or UTP, which is labeled with a marker probe. 
     
     
       6. A method of screening for inhibitors of RdRp, comprising the following steps:
 a) incubating a candidate compound with the composition according to  claim 3 ; and 
 b) determining whether the candidate compound promotes or inhibits an RNA synthesis activity of SARS-CoV nsp12. 
 
     
     
       7. The method of  claim 6 , wherein the step b) comprises measuring whether an RNA labeled with a marker nucleotide is synthesized or not.